Method for electrical detection of a binding reaction

ABSTRACT

A method is disclosed for detecting the occurrence of a binding or complex-forming reaction between specific substances by utilizing the binding reaction to modify an electrical circuit, and then measuring a change in the electrical state of the circuit. A diagnostic element useful in such a method includes a layer of a biogenic substance, such as an antigen, coated onto a non-conductive base between a pair of electrical conductors superposed on the base. Antibodies which react with the antigen are treated so that they become bound to particles. The particles having antibody bound thereto are then added to is the antigen lay e base and allowed to react therewith. The particles are thereby bound to the base due to the binding reaction between the antigen and antibody to thereby form aggregates of electrically conductive particles which modify the circuit. The particles are then selectively coated with a conductive substance. The method of the invention is highly useful for the detection of antigens and antibodies in the blood serum of a human patient.

This is a continuation of U.S. Ser. No. 07/590,599 filed Sep. 2, 1990,now U.S. Pat. No. 137,827, which is a continuation of U.S. Ser. No.07/269,971 filed Nov. 10, 1987, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 06,843,982 filed Mar. 25, 1986,now U.S. Pat. No. 4,794,089.

This invention was made with government support under a small businessinnovation research grant awarded by the National Institute of GeneralMedical Sciences. The government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a diagnostic element for electricallydetecting a binding reaction between a pair of chemical substances,particularly biogenic substances such as antigens and antibodies ornucleotides. The invention further concerns a new immunoassay method.

BACKGROUND OF THE INVENTION

It is well known that when a foreign substance such as a bacterium orvirus enters a human or animal body, production of antibodies to combatthe infection is stimulated by the presence of one or more antigens.Antigens related to invading organisms comprise a foreign substance thatcomes from the invading organism, such as a piece of bacterium or virus.More generally, an antigen is any substance capable of triggering animmune response. Certain specialized cells of the body contact anantigen and produce antibodies specifically designed for that antigen.When released in the body, such antibodies identify and bind to theantigen, thereby combating the infection. Antibodies are highly specificand will generally bind only to the antigen which stimulated theircreation.

When a person has been infected with certain diseases, that person'sblood will often contain measurable levels of antigen specific to thatdisease. To determine whether such an infection is present, animmunodiagnostic test is performed using a sample of the patient'sblood. The sample is mixed with a solution known to contain antibodiesspecific to a certain disease or condition. If an antigen-antibodyreaction occurs, the test result is positive, and the antigen isdetected. Such a test is typically reversible, i.e., a solution orreagent known to contain a certain antigen can be used to determinewhether or not the corresponding antibody is present in a sample.However, the antigen-antibody reaction occurs on a microscopic level andis not readily observable. Thus, all known immunodiagnostic testsprovide some type of means for indicating that the antigen-antibodyreaction has occurred.

A variety of techniques have been used to detect antigen-antibodyreactions. The principal techniques presently in use are enzymeimmunoassay, immunofluorescence, and radioimmunoassay. In typical enzymeimmunoassay procedures, the antigen-antibody reaction is detected by theformation, by an enzyme, of a colored product from a colorlesssubstrate. Immunofluorescence techniques indicate that a reaction hasoccurred by emission of small quantities of light which must generallybe observed microscopically. Radioimmunoassay utilizes radioactivelabeling substances so that occurrence of the antigen-antibody reactionis measured by the presence or absence of small amounts ofradioactivity. These known methods are reliable but are slow andtedious.

Recently several types of electrical immunoassay techniques have beendeveloped. One such technique utilizes field effect transistors coatedwith a layer of antibody in the gate region. If an antigen-antibodyreaction occurs, the charge concentration of the transistor changes.Examples of this type of system are given in Schenck U.S. Pat. No.4,238,757, issued Dec. 9, 1980; Guckel U.S. Pat. No. 4,180,771, issuedDec. 25, 1981; Malmros U.S. Pat. Nos. 4,334,880, issued Jun. 15, 1982and 4,444,892, issued Apr. 24, 1984, and Japanese Patent Publication No.60-29658. In another system, a body fluid containing an analyte to bedetected is deposited onto a surface which has been coated with areagent that binds specifically to the analyte, so that a bindingreaction takes place. A tagged reagent is then added which reacts withthe analyte-reagent complex or with the reagent to change the electricalreactance of the surface. See Ebersole U.S. Pat. No. 4,219,335, issuedAug. 26, 1980.

Several other methods have been proposed for measuring immunologicreactions electrically. A voltametric immunoassay can be carried bylabeling one immunoreactant with an electroactive substance. Pace U.S.Pat. No. 4,233,144, issued Nov. 11, 1980, is illustrative of one suchtechnique. Another method involves sandwiching an antigen-antibody layerbetween two conductive layers and measuring the electrical capacitanceof the resulting laminate. Giaever U.S. Pat. No. 4,054,646, issued Oct.18, 1977, describes such a method. A further type ofcapacitance-measuring system includes a pair of electrodes coated with asubstrate and immersed in a medium containing a material whichspecifically binds with the substrate, as described in Arwin U.S. Pat.No. 4,072,576. A further method combines change effect signal detectionwith an enzyme immunoassay technique. Such a method is disclosed byGibbons U.S. Pat. No. 4,287,300, issued Sep. 1, 1981. The foregoingelectrical methods have, however, failed to provide medicalpractitioners and laboratories with a simple, fast, sensitive,inexpensive and easy-to-use method of performing an immunodiagnostictest.

One aspect of the present invention involves the use of antigen orantibody-labeled colloidal gold particles. In general, "colloidal gold"refers to a suspension of fine gold particles in water or aqueoussolution. Preparation of such particles is disclosed by DeMey, et al.U.S. Pat. No. 4,446,238, issued May 1, 1984, and DeMey, et al. U.S. Pat.No. 4,420,558, issued Dec. 13, 1983. The entire contents of both suchDeMey patents are incorporated herein by reference. Such colloidal goldpreparations have been previously used in immunodiagnostic tests whereinthe results are determined optically by observing small amounts of lightreflected as a result of the antigen-antibody reaction. The foregoingpatents to DeMey disclose a bright field light method of the foregoingtype. Silver enhancement has been previously used as a means forstaining gold particles. The present invention advantageously employscolloidal gold, optionally with silver enhancement, in a newimmunodiagnostic method.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting a binding reactionbetween a pair of first and second substances, particularly biogenicsubstances, which specifically bind together. The method of theinvention involves bringing the substances together so that the bindingreaction between them causes full or partial completion (closing) of anessentially open electrical circuit. The resulting change in theelectrical state of the circuit indicates the binding reaction.

According to a further aspect of the invention, a diagnostic element foruse in detecting a binding reaction comprises a pair of spaced-apart,electrical conductors, particularly conductive layers, disposed on asubstantially non-electrically conductive base. The base may comprise asupport having a layer formed thereon which has a high affinity forprotein binding and has a moderate to high resistance in comparison tothe conductors. The space between the conductors defines a path orchannel. One of a pair of substances which bind to each other isdeposited on and affixed to the binding layer of the non-conductive basebetween the conductors, such as on the bottom wall of a channel. Meansforming an electrical circuit is connected to each of the conductors sothat the channel constitutes a break in the circuit.

As used herein, the term "diagnostic element" refers to the base,conductors, and layer of one of the binding substances, without themeans defining the electrical circuit. Such a diagnostic element andmeans forming an electrical circuit can readily be used in conjunctionwith any suitable means for fully or partially bridging the break in thecircuit due to the binding reaction between the pair of substances. Onesuch means involves adhering one of the substances to the surfaces ofelectrically conductive particles.

BRIEF DESCRIPTION OF THE DRAWING

Preferred exemplary embodiments will hereafter be described inconjunction with the appended drawing, wherein like designations denotelike elements, and:

FIGS. 1A, 1B, 1C, 1D and 1E and FIGS. 2A, 2B, 2C, 2D and 2E areschematic diagrams illustrating an immunodiagnostic method according tothe invention;

FIG. 3 is a schematic diagram illustrating a reaction detector accordingto one embodiment of the invention;

FIGS. 4A and 4B are schematic diagrams showing aggregate formationaccording to the method of FIGS. 1A-1E and 2A-2E, respectively;

FIG. 5 is a schematic diagram showing binding of a conductive particleto a non-conductive base according to one embodiment of the invention;

FIG. 6 is a cross-sectional view of a diagnostic element according toone embodiment of the invention;

FIG. 7 is a cross-sectional view of an alternative diagnostic elementaccording to the invention;

FIG. 8 is a plan view of a multiple diagnostic element according to theinvention;

FIG. 9 is a plan view of an alternative multiple diagnostic elementaccording to the invention;

FIG. 10 is a schematic diagram of an uncoated bound aggregate accordingto the invention;

FIG. 11 is a schematic diagram of a coated bound aggregate according tothe invention;

FIG. 12 is a schematic diagram of resistive shunting of currentaccording to the invention;

FIG. 13 is a graph wherein resistance values after silver overcoatingand silver counts, both as obtained in Example 4, are plotted againstgold-antibody conjugate dilution; and

FIG. 14 is a graph wherein percent of maximum resistance determined inExample 5 is plotted against amount of competing antigen.

DETAILED DESCRIPTION

The method of the invention is particularly useful for detectingantigens in the fluids or tissues of humans or animals. Such antigensinclude drugs, toxins, hormones, allergens, tumor markers, factors,enzymes" steroids, nucleotides and other substances as listed in HuangU.S. Pat. No. 4,327,073, issued Apr. 27, 1982, the entire content ofwhich is incorporated herein by reference. Any of the foregoing listedsubstances can provoke the production of a reactive substance (antibody)which reacts with and binds to the antigen. Accordingly, the method ofthe present invention is useful for detection of a wide variety ofsubstances which may be present in the living body of a human or loweranimal, for example, in drug overdose treatment, where it is desired toquickly determine which drug a patient has taken.

FIGS. 1A-1E schematically illustrate a method for detecting an antigenaccording to the present invention. Referring to FIGS. 1A through 1C, apatient sample 11A (FIG. 1A) such as whole blood, blood serum or urine,containing a particular antigen 12A is mixed with a colloidal goldpreparation 13A (FIG. 1B) containing a predetermined amount of goldparticles 14A having antibodies 15A fixed to the outer surfaces thereof.Antibodies 15A specifically bind to antigen 12A (FIG. 1C). In theresulting mixture 16A, antigen 12A binds with available antibodies 15A,resulting in free complexes 18A, comprising both antigen 12A andantibody 15A bound to particles 14A. Since there are more antibodies 15Athan antigens 12A, some antibodies 15A remain free, i.e., unbound to anantigen 12A.

As illustrated in FIGS. 2A-2C, the foregoing procedure is also carriedout using a control sample 11B (FIG. 2) lacking the antigen 12A and asecond colloidal gold preparation 13B (FIG. 2B) substantially identicalin composition to preparation 13A used with patient sample 11A. Thethus-formed second mixture 16B (FIG. 2C) lacks the complexes 18A shownin FIG. 1A, and correspondingly has a greater number of particles 14Bhaving unbound antigen 15B on the surfaces thereof. The first mixture16A, corresponding to the patient sample (FIG. 1C), and the secondmixture 16B (FIG. 2C), corresponding to the control, are then ready foruse with a corresponding reaction detector 20A, 20B according to theinvention (FIGS. 1D, 2D).

Referring now to FIG. 3, reaction detector 20 includes anon-electrically conductive base 22, a pair of thin, spaced-apartelectrically conductive layers 23, 24 disposed side-by-side on base 22with a channel 32 formed therebetween, and means defining an electricalcircuit, such as an ohmmeter 26 functionally connected to layers 23, 24as shown by means such as wires 28. Layers 23, 24 act as a pair ofpositive and negative terminals for the circuit.

Referring again to FIGS. 1 and 2, identical first and second reactiondetectors 20A (FIG. 1D) and 20B (FIG. 2D) are prepared in advance foruse with mixtures 16A, 16B, respectively. Samples of antigen in acarrier liquid (e.g., water or saline solution) are poured into shallowchannels or grooves 32A, 32B defined between layers 23A,B and 24A,B tocause antigen to bind to the surfaces of bottom walls 33A,B of channels32A,B to form antigen layers 30A, 30B (FIGS. 1D, 2D). These antigenlayers 30A, 30B are made of the same type of antigen as antigen 12A tobe detected.

Referring now to FIG. 1E, first mixture 16A (FIG. 1C), corresponding topatient sample 11A (FIG. 1A), is poured into channel 32A of firstdetector 20A to cause binding of antigen layer 30A and antibody 15A. Theforegoing procedure is also carried out using the control mixture 16B(lacking complexes 18A) and second detector 20B (FIG. 2E). Conductiveparticles 14A having free antibodies 15A thereon effectively becomebound to bottom wall 33A via antigen layer 30A due to theantigen-antibody binding reaction. Complexes 18A containing antigen 12Afrom sample 11A do not tend to become bound to bottom wall 33A.

After a suitable time to allow the antigen-antibody interaction to takeplace, channel 32A is flushed with a suitable liquid, e.g., water orsaline solution, to wash away any unbound particles 14A, then dried byany suitable means, such as heating or allowing the reaction detector tostand open to the air. The resistance measurement can also be performedwet, without any drying step. Even when wet, the difference in measuredresistance is sufficient to indicate whether the binding reaction hasoccurred.

Non-specific binding of particles to layers 30A, 30B occurs to someextent. Ordinary washing procedures may not be sufficient to remove suchnon-specifically bound particles from the antigen layer. However, it hasbeen found that ultrasonic treatment of the samples at a low level canremove non-specifically bound particles without removing specificallybound particles, i.e., particles bound due to antigen-antibody bindinginvolving the substance being detected for. This is particularlyimportant as a means of preventing a false positive result. Such a falsepositive result is a decrease in resistance due to non-specificallybound particles, rather than specifically bound particles.

Referring now to FIGS. 4A and 4B, FIG. 4A corresponds to the same stateas FIG. 1E, and FIG. 4B corresponds to the same state as FIG. 2E. FIGS.4A, 4B illustrate the differences in the extent of the binding reactionnot apparent in FIGS. 1E, 2E. Ohmmeters 26A and 26B register theresistance across channels 32A and 32B for each of the detectors 20A and20B. For the control (FIG. 4B), all particles 14B having free antibodies15B deposited thereon are available for binding with antigen layer 30Bbound to bottom wall 33B. As a result, complexes 35B are formed atbottom wall 33B and are anchored thereto. As illustrated in FIG. 4B,complexes 35B tend to cluster together in contact with each other toform aggregates or chains 39B which effectively bridge channel 32B.Since particles 14B are electrically conductive, aggregates 39Beffectively provide an electrical connection between layers 23B and 24B,completing an electrical circuit defined by ohmmeter 26B, wires 28B,layers 23B, 24B, and aggregates 39B. This is reflected by the resistancereading given by ohmmeter 26B. A drastic decrease in resistance occursas a result of bridging of aggregates 39B.

The reaction for the mixture 16A corresponding to the patient sampleproceeds in a similar fashion, except that this mixture 16A alreadycontains complexes 18A formed by reaction of antigen 12A with antibody15A. Since the antibodies of these complexes 18A are already bound withat least some antigen 12A, these complexes 18A do not tend to bind tothe layer of antigen 30A at bottom wall 33A. In mixture 16A, the numberof free antibodies 15A deposited on particles 14A is less than inmixture 16B, since some of these antibodies 15A were used to formcomplexes 18A. As shown in FIG. 4A, aggregates 39A form, but there arefewer such aggregates, and correspondingly less bridging of channel 32A.As a result, the decrease in resistance registered by ohmmeter 26A, ifany, is less than the decrease in resistance registered by ohmmeter 26B.This difference in readings indicates the presence cf antigen 12A in thepatient sample 11A. If patient sample 11A does not contain any antigen12A, then the decrease in resistance for reaction detector 20A would bethe same as the decrease in resistance for detector 20B.

If resistance values corresponding to specific antigen levels in thesample are well known for a specific test, the foregoing procedure canbe carried out without the control illustrated in FIGS. 2A-2E, 4B.However, the use of a control is preferred because the comparativeresistance readings produced by the control afford more accurateresults.

The procedure shown in FIGS. 1 through 4 is greatly simplified forpurposes of illustration. Conductive particles 14A,B are larger thanantigen 12A and antibodies 15A,B. A number of antibodies 15A,B are boundto a single conductive particle 14A or 14B, and similarly a number ofantigens 12A can bind with antibodies 15A on the surface of a singleparticle 14A or 14B. FIG. 5 schematically illustrates how a conductiveparticle 14B having antibodies 15B on its surface becomes bound tobottom wall 33B via antigen layer 30B.

The method of FIGS. 1-4 utilizes a positive control, that is, a decreasein resistance due to bridging of the channel by the conductive particlesoccurs for the control sample, not the unknown (patient) sample. Themethod of the invention may also be carried out using a negative controlwherein the sample containing the unknown, for example, an antibody, isreacted directly with the gold particles, and then the gold particlesare brought in contact with the reaction surface coated with thecorresponding antigen. The procedure is substantially the same as shownin FIG. 2B, except that the amount of the antibody is unknown.

Many variations of the method of the invention, including bothcompetitive and non-competitive procedures, are possible. The processillustrated in FIGS. 1A-1E is a competitive reaction wherein the antigenis bound to the diagnostic element, and free antigen in the sample 11Acompetes with the bound antigen for sites on the conductively labelledantibody. The preincubation step shown in FIG. 1C may be omitted, andthe sample 11A and colloidal gold 13A may be added directly to antigenlayer 30A bound to the diagnostic element. Alternatively, the antibodymay instead be bound to the diagnostic element, and conductivelylabelled antigen can compete with free antigen in the sample forantibody binding sites. In either case, the resistance measurements aredirectly related to the amount of free antigen in the sample, in otherwords, the measured resistance increases directly with increasingamounts of free antigen in the sample.

In a non-competitive variation useful for antigens that can bind to morethan one antibody at the same time, a first antibody is bound to thediagnostic element in a predetermined amount in excess of the amountrequired to bind all available free antigen in the sample. The sample isadded to the bound antibody and allowed to react. A second, conductivelylabelled antibody is added, either later or at the same time as thesample. The second antibody also reacts with the antigen, resulting in acomplex comprising first antibody-antigen-second antibody-conductiveparticle bound to the diagnostic element. The two binding sites on theantigen may be identical, structurally different, or two-siteimmunometric. The resulting resistance measurements are inverselyrelated to the amounts of free antigen in the sample, such that theresistance decreases as the amount of free antigen increases.

Another non-competitive variation is useful for determining amounts ofspecific antibody in a sample, particularly an antibody titer for adisease or allergy. The antigen is bound to the diagnostic element insufficient excess to bind to the antibody in proportion to itsconcentration. A sample containing free antibody is added to the boundantigen and allowed to react. A secondary antibody carrying theconductive particles is added, either later or at the same time as thesample. The secondary antibody reacts with the first antibody, i.e.,treats it as an antigen, forming a complex comprising antigen-firstantibody-second antibody-conductive particle. The secondary antibodycould be, for example, anti-immunoglobulin G or E. The resultingresistance measurements are inversely related to the amount of free(first) antibody in the sample. A secondary antibody can also be used inembodiments wherein resistance change is directly related to measuredantigen in the sample in order to increase sensitivity.

Bases 22A, 22B discussed above must have a highly bioreactive surface."Bioreactive" as discussed herein refers to the ability of the surfaceto bind biogenic substances such as proteins or nucleotides.Bioreactivity values measured for various organic and inorganicsubstances vary widely even among chemically similar substances.However, certain plastics and metal oxides and nitrides are generallybioreactive. In a series of enzyme immunoassay experiments (see Example3) the absorption of light at 490 nanometers was used as a standard fordetermining binding by a rabbit IgG-goat anti-rabbit IgG complex to eachsurface tested. Nylon 66 had the highest level of bioreactivitymeasured. For purposes of the present invention, substances having abioreactivity at least 70% that of Nylon 66 for proteins are consideredhighly bioreactive. Compounds having about 50-70% as much bioreactivityas Nylon 66 are mildly bioreactive, compounds having 10-50% are somewhatbioreactive, and compositions having a bioreactivity of about 10% orless are bioinert.

It has been found that the following substances are highly bioreactivewith proteins: Nylon 66, polypropylene, mylar, chromium oxide, phenolicplastic, polystyrene, and vinyl. Chromium oxide is unusually high inprotein bioreactivity for a metal oxide. Moderately bioreactivesubstances for proteins include chromium, titanium oxynitride, nickeloxide, tantalum nitride and carbon. Poorly bioreactive substances forproteins include titanium oxide, boron nitride, and silicon oxide.Bioinert substances for proteins include silicon nitride, bariumtitanium oxide, indium tin oxide, aluminum oxide and glass.

FIGS. 6 and 7 illustrate two diagnostic elements 10A, 10B that can beused in the method of the invention. In FIG. 7, diagnostic element 10Aaccording to the invention includes a support 40 such as a glass plate,e.g. a microscope slide, coated with a thin layer 45A of an electricallyresistive, bioreactive substance such as chromium oxide. A pair ofconductive layers, such as chromium metal layers 51, 52, are superposedon the resistive, bioreactive layer 45A. A channel 42 separates layers51, 52. A layer of an antigen 53 spans channel 42, and may also coverlayers 51, 52. Layers 45A, 51, 52 are preferably formed by sputtering ona glass plate. Channel 42 is then formed by photolithography using anetchant that selectively attacks the conductive layer but does notattack layer 45A. In this diagnostic element, the width of channel 42relative to the size of the conductive particles to be used is importantbecause the binding reaction is primarily indicated by complete bridgingof channel 42 by aggregates of the conductive particles. The resistivelayer 45A is highly resistive, i.e. more than about 10⁶ ohms-cm, so thatvirtually no change in resistance is caused by partial bridging as shownin FIG. 4A.

FIG. 6 illustrates an alternative diagnostic element wherein conductivelayers 51, 52 are formed directly on support 40, with channel 42 formedtherebetween. Resistive layer 45B is superposed over support 40 andlayers 51, 52, and antigen layer 53 is then formed thereon. In thisembodiment layer 45B is only moderately resistive, e.g. 10³ -10⁶ohms-cm, so that a substantial change in resistance will occur due toresistive shunting of current through aggregates bound to layer 53, evenwhen no complete bridging occurs.

FIG. 12 illustrates a resistor through which resistive shunting occurs.In seeking the path 71 of least resistance, the current will be shuntedthrough bound aggregates 72 which offer a lower resistance than thesupport 40. The overall resistance drop across channel 42 will depend onhow many aggregates are bound, and can provide a quantitative indicationof the amount of binding being measured. In the resistor shown, thecalculated resistance drops from 70,000 to 40,030 ohms following thereaction.

The width of channel 32 or 42 may vary, particularly in relation to thesimple numerical average diameter of the conductive particles forming achain to bridge the channel. The following table states preferred rangesfor dimensions for gap-bridging embodiments according to the presentinvention:

                  TABLE 1                                                         ______________________________________                                        Average Particle                                                                            Channel    Ratio of Channel                                     Diameter      Width      Width to Particle                                    (Microns)     (Microns)  Diameter                                             ______________________________________                                        0.01-500        0.1-20,000                                                                              5:1 to 40:1                                         0.01-10        0.1-100   10:1 to 30:1                                         0.01-1         1-25      15:1 to 25:1                                         ______________________________________                                    

A 20:1 ratio of channel width to average particle diameter is typical,e.g., the channel has a width of 10 microns, and the average diameter ofthe conductive particles is 0.5 microns. The foregoing ranges are alsouseful in resistive shunting embodiments, but much larger channel widthsmay be employed, such as up to 1 mm 1 cm, or greater, depending on thedesired application.

As an alternative to a channel, diagnostic elements according to theinvention, especially resistive shunting embodiments, may utilize aresistive, bioreactive path other than a channel. Such a path maycomprise, for example, a curved line which spans the conductors but doesnot represent the shortest distance between them. Similarly, theconductors need not be in the form of layers. Small wires superposed onthe base can, for example, be employed as the conductors.

Proteins have an affinity for materials such as polystyrene, chromiumoxide and the like, and tend to become bound thereto under suitableconditions. Proteins can also readily become bound to the surfaces offine metal particles, such as gold particles, using the proceduredescribed below. In embodiments of the invention which involveantigen-antibody binding, it is preferred to bind the antigen to thebottom wall of the channel and bind the antibodies to the conductiveparticles. However, the reverse arrangement (antigenparticles,antibody-channel) can also be employed.

Conductive layers 51, 52 may have any desired dimensions which provefunctional. To reduce the size of the diagnostic element, these layersare generally as thin as possible, and preferably have a thickness nogreater than about 5 microns, preferably no greater than about 0.5microns, particularly a thickness in the range of 0.001-0.005 microns.Conventional sputter deposition can be readily used to form theconductive layers in any desired shape.

Layers 51, 52 (or 23, 24) may be formed of any suitable conductivematerial, particularly an electrically conductive metal such as gold,silver, platinum, copper, chromium or aluminum. Particles 14A, 14B arepreferably made from a conductive metal such as gold, silver, orplatinum, and may also be made of carbon platelets or plastic particleshaving a conduct live metal coating, especially gold-coated polystyrenespheres. Such coated spheres are lighter than comparable solid metalspheres and are thus better able to maintain bonding to the surface.

The extent of binding of an antibody to metal particles is influenced byvarious factors. A series of enzyme immunoassay experiments demonstratedthat affinity-purified antibody tends to bind to gold particles to amuch greater extent than impure, whole serum antibody, and that the pHof the system also had a strong effect. A slightly acidic pH (e.g. 6-7)produced several-fold greater binding than a basic pH of 9-10 for large(greater than 0.5 μm diameter) particles. Polyethylene glycol treatmentof the gold particles may also be used if needed to stabilize large goldparticles; see Horisberger et al., J. Histochem. Cytochem., 25:295-305(1977).

The antibody concentration on the gold particles should be high enoughto allow the antigen-antibody reaction to occur on the surfaces of thegold particles to an extent sufficient for detection by the method ofthe invention. On the other hand, if the antibody concentration is toogreat, the antibody layer may have an insulating effect which willresult in a false negative result, i.e., will block the drop inresistance that would normally occur, unless an additional step ofovercoating the aggregates with a conductive metal is used, as describedhereafter.

Base 22 described above is made of a bioreactive plastic, preferablypolypropylene, mylar, phenolic plastic, vinyl, methyl cellulose, nylonor polystyrene. The polarity of protein molecules causes such moleculesto bind to such a plastic base to form a substantially complete,homogenous coating of antigen. Glass by itself is not generally employedas the base (support) since antigens have a poor affinity for a glasssurface and it has proved difficult to adhere a layer of antigen to aglass slide. However, according to a further aspect of the invention, ithas been found that a glass slide can be surface treated so that antigenaffinity for the coating on the glass slide becomes as great or greaterthan antigen affinity for a conventional polystyrene slide. As notedabove, this surface treatment comprises coating the glass support 40with a thin layer 45A or 45B of a material having a moderate to highelectrical resistance as compared to the conductive layers. Theresistance of this layer is in the range 10³ -10⁸ ohms-cm, preferably10⁴ -10⁷ ohms-cm, as compared to a resistance of less than 100 ohms-cmfor the conductors. Moderately resistive materials for purposes of theinvention are those having measured resistances in the range of 10³ upto 10⁶ ohms-cm when deposited on a glass support, whereas highlyresistive materials have comparable measured resistances of 10⁶ or more.

Bioreactive substances suitable for forming layer 45A or 45B includecarbon, hydrophilic organic polymers, and inorganic metal oxides andnitrides. Such inorganic metal compounds include oxides, nitrides andoxynitrides of boron, aiuminum, silicon, cadmium, copper, nickel,cobalt, iron, manganese, and metals of Groups IIA, IIIB through VIB.Especially preferred inorganic metal compounds include chromium oxide(CrO₃), titanium oxynitride (TiO_(x) N_(y)), tantalum nitride, cermetmaterials such as chromium/silicon oxide (Cr·Sio) or gold/silicon oxide,and other inorganic materials currently used in resistors. Hydrophilicorganic polymers include well-known plastics such as mylar, polystyreneand nylons, such as Nylon 66.

If chromium oxide is used as layer 45A, it is usually deposited in theform of CrO₂. To make it more bioreactive, the layer may be aged in thepresence of oxygen and water vapor to convert it to CrO₃, which ishydrophilic. This aging can be accelerated by placing the coated base orfinished diagnostic element in a humidity chamber. Carbon in anyconvenient form, e.g. graphite, is a theoretically ideal material forlayer 45 in view of its biocompatability, although its thin filmresistance may be hard to control within narrow ranges.

Cermet resistor materials have a high resistance, are stable, and havelow negative temperature coefficients, such that the diagnostic elementwill not be excessively temperature sensitive. When heated in air,cermet films oxidize and increase in resistance, and are usuallyprovided with a protective overcoating to prevent oxidation and/orhydration. In the present invention, however, layer 45 does not requiresuch a protective coating.

FIG. 8 illustrates a further embodiment of the diagnostic element of theinvention wherein a multiplicity of reaction sites are disposed on asingle non-conductive base 22. Each reaction site comprises a pair ofconductors, such as layers 23, 24, having a layer of a biogenicsubstance which undergoes specific binding disposed in the channel orpath therebetween. The biogenic substance, such as an antigen, may bethe same or different for each reaction site. Conductive means 28 forthis embodiment comprises a series of individual electrical conductors61 which each connect to a common conductor 62, which in turn connectsto a terminal plate 63 mounted on the edge of base 22. Pairs of layers23, 24 are arrayed in rows and columns on base 22.

In the embodiment of FIG. 8, rows of plates 63 are disposed on adjacentsides of rectangular base 22. To prevent overlapping between a first set66 of conductors 62 connected to layers 23 and a second set 67 ofconductors 62 connected to layers 24, all but one of conductors 62 ofsecond set 67 are located on the reverse side of base 22. Theseconductors 62 are illustrated by broken lines in FIG. 8. Outer conductor62A does not cross any conductors 62 of first set 66 and thus does notneed to be on the other side of base 22, although it can be so locatedif desired. Individual conductors 61 connected to common conductors 62of second set 67 on the reverse side of base 22 include portions 69which extend through the thickness of base 22 and connect with suchconductors 62, shown by broken lines in FIG. 8.

Ohmmeter 26 can be connected to various combinations of terminal plates63 to measure resistance for each pair of layers 23, 24. To make such ameasurement, ohmmeter 26 is connected to two plates 63 on differentedges of base 22. Connecting an ohmmeter to plates 63C, 63D would testthe indicated pair of layers 23C, 24D. This embodiment allows a singlediagnostic element according to the invention to test a single patientsample for a number of different substances, such as antigens, sinceeach pair of layers 23, 24 can have a different substance boundtherebetween, or have no substance bound therebetween so as to provide acontrol. In the alternative, the same substance may be tested for anumber of times to provide a more certain result.

In an alternative embodiment shown in FIG. 9, terminals 63 are disposedalong only one edge of base 22. One terminal 63E is connected to eachlayer 23 by a single, multibranched conductor 62E. A series of terminals63F are each connected to one of layers 24 by separate conductors 62F.Conductors 62E,F are arranged to not cross each other, so that all ofconductors 62E,F are disposed on the front face of the diagnosticelement as shown. For this purpose terminal 63E is located at one end ofthe row of spaced-apart terminals 63E,F. This embodiment avoids the needto provide wires, conductive lines, or the like on both sides of theplate, and allows the terminals to be disposed on a common edge.

Resistive shunting according to the method of the invention may begreatly enhanced by overcoating the aggregates with a layer of aconductive substance, particularly a metal such as silver, gold, orplatinum. The applied coating must stick selectively to the aggregatesbut not to the remainder of the path between the conductors. If goldparticles are used as the particles to which one of the bindingsubstances are bound, silver enhancement may be used to form aconductive silver coating over the aggregates.

Conductive metal overcoating is especially useful for resistive shuntingembodiments of the invention for two reasons. First, it can eliminateproblems with junction resistance effects. Referring to FIG. 10,resistive shunting is minimized by the relatively small contact surfaceof the aggregates 76 with the underlying layer 53. In addition, thebiogenic coating 77 on the gold particles 78 can form an insulatingbarrier such that the junction resistance R_(j) is high enough toprevent. Resistive shunting. By contrast, the conductive metal coating79 shown in FIG. 11 creates a much lower resistance and allows resistiveshunting to occur. This embodiment of the invention is especiallypreferred because it improves reliability, as demonstrated by theresults of Example 2 below.

Second, metal overcoating can eliminate the need to use particles whichare both bioreactive and conductive. Since the metal overcoating canconduct the current, the particles can be made of a non-conductivesubstance, e.g., non-conductive beads may be used so long as the metalovercoating sticks selectively thereto. This advantage also pertains togap bridging embodiments as well.

The following examples describe a gap bridging embodiment (Example 1), aresistive shunting embodiment (Example 2) and an example of a procedurefor determining bioreactivity according to the invention (Example 3).

EXAMPLE 1 A. Preparation of Colloidal Gold

The following procedure was used to make a suspension of gold particles.Glassware including a flask and stirring bar were cleaned by sonicationin a bath sonicator, first in ethanol, then acetone, then petroleumether, for about 5 minutes each. The glassware was then blown dry with aFreon refrigerant, then rinsed twice with double glass-distilled water.About 1 ml (milliliter) of a 1% w/v (weight/volume) filtered solution ofgold chloride in water and 99 ml of filtered, double glass distilledwater were added to the cleaned flask. The resulting mixture was thenheated and stirred with the cleaned stirring bar until boiling. At thattime 300 μl of a 1% w/v filtered solution of trisodium citrate in waterwas added. Boiling was continued with stirring, and additional 1%trisodium citrate solution was added in amounts of 100, 50 and 50 μl atintervals of 5, 12 and 15 minutes, respectively, from the time themixture began boiling. After 30 minutes of boiling the flask was removedfrom heat and allowed to cool. The average particle size of theresulting colloidal gold particles was 100 nm as determined using atransmission electron microscope. The flask containing the colloidalgold preparation was sealed and stored at 4° C.

B. Preparation of Gold-Labelled Proteins

About 9 ml of the colloidal gold preparation made in part A above, 100μl of 0.2 M potassium carbonate and 20 μl of a protein, anaffinity-purified goat anti-rabbit immunoglobulin (IgG) antibody, wereadded to a 15 ml conical centrifuge tube. The ingredients were mixedovernight on a tube rotator. 1 ml of a filtered aqueous polyethyleneglycol solution (2 mg PEG/ml) was then added as a stabilizing agent.Filtration in this and subsequent procedures was carried out using a 0.2micron filter disk fitted into a syringe. The resulting gold-proteinconjugates were concentrated by centrifugation at 11,000 rpm for 10minutes. The supernatant was discarded, and the resulting pellet of goldparticles was pipetted into a 1.5 ml centrifuge tube. The particles werethen washed by centrifugation four times with a phosphate bufferedsaline solution (0.01 M sodium phosphate dibasic, 0.15 M sodiumchloride, 0.1 mg/ml polyethylene glycol, pH 7.4) containing 0.2 % sodiumazide as a preservative to remove unbound protein. The conjugated goldparticles were then resuspended in 1.4 ml of phosphate buffered salinesolution containing 0.2 % sodium azide.

C. Preparation of Diagnostic Element

A glass slide was coated with a thin layer of chromium oxide (CrO_(x),wherein x=1-3) by radiofrequency (rf) magnetron sputter deposition usinga Materials Research Corporation Model 822 Sputtersphere. A chromiumplate target was used as the cathode. The glass slide was placed on acopper plate anode disposed beneath the cathode. Deposition was carriedout under the following conditions: system base pressure 3×10⁻⁷ Torr,total pressure 8×10⁻³ Torr, forward power 1,OOO W, target voltage 335 V,reflected power 0 watts, cathode to anode distance 21/2inches, and asputtering time of 30 minutes. The system was backfilled with oxygen.The desired chromium oxide layer having a thickness of about 0.1 μm wasthereby formed by deposition in the oxygen plasma. A layer of chromiumwas then deposited over the chromium oxide layer by repeating theforegoing procedure in an inert argon plasma, resulting in a surfacelayer of chromium having a thickness of 1 μm.

D. Photolithography

A line having a width of 5 μm was then formed in the surface chromiumlayer of the double-coated slide of part C by photolithography. Inyellow room light, a layer of photoresist plastic was first formed overthe chromium layer as follows. The twice-coated slide was placed in thechuck of a Headway Model EC101 spinner under vacuum. About 3 ml ofShipley 111 S photoresist was applied over the chromium layer andallowed to spread for 10 seconds . The slide was then spun at 2000 rpmfor 30 seconds to form a uniform 1 μm thick photoresist coating. Severalsuch photoresist-coated slides were then baked in a hot plate oven on arack for about 20 minutes at 80° C., then allowed to cool for 5 minutes.

A Suss MJB55 photolithographic mask aligner was calibrated and preparedfor use. A mask defining a 5 μm line was cleaned thoroughly withdissolved soap and water, then dried with a nitrogen stream that left noresidue. The mask was then placed in a mask holder under vacuum, and themask holder was then placed in position on the aligner. Thetriple-coated slide was placed beneath the 5 μm line of the mask. Themask holder was then locked in place, and the aligner was used to exposethe photoresist layer to UV light for 18 seconds. The exposed slide wasthen removed from the aligner.

The slides were then immersed in a fresh 4:1 developer solution (4 partswater to 1 part Shipley 303A developer) for 2 minutes with no agitation,and then promptly rinsed with distilled water. Each slide was thenimmersed in the etchant, a solution heated to 45° C. consisting of 90.8g AlCl₃, 27.0 g ZnCl₂, 6 ml H₃ PO₄ and 80 ml distilled water, for about10 seconds . During this time the etchant removed undevelopedphotoresist material and chromium metal along the 5 micron line whichwas previously exposed to UV light. However, the etchant did not attackthe chromium oxide layer. After etching, the slide was promptly rinsedin distilled water. The photoresist material was then removed from thesurface of the chromium layers by immersing the slides for about 5minutes in Shipley 1112A remover. Thereafter the slide was rinsedthoroughly in tap water, then rinsed ultrasonically in distilled waterfor 10 minutes. The slide was then re-immersed in the remover, and therinsing steps were repeated to ensure that the photoresist material wascompletely removed.

E. Measurement of Binding Reaction

The resistance of the diagnostic elements prepared in part D wasmeasured using an ohmmeter. Each element was then placed in a test tubeand rinsed with a coating buffer (0.015 M sodium carbonate, 0.035 Msodium bicarbonate, 0.003 M sodium azide, pH 9.8). Pairs of diagnosticelements were prepared by adding either rabbit immunoglobulin (IgG) orbovine serum albumin (BSA) to each test tube. The elements wereincubated by allowing the tubes to stand overnight at room temperature.The protein-coated elements were then washed three times with aPBS-Triton buffer (phosuhate buffered saline solution as described abovecontaining 0.1 vol. % Triton X-100 as a wetting agent) to remove excessunbound protein. The coated substrates were then placed on a piece ofparafilm in a petri dish. The colloidal gold-protein conjugate was thenapplied to each element in an amount sufficient to cover the uppersurface of each element, and the elements were allowed to incubate(stand) for 20 minutes at room temperature. Each element was then washedby aspiration once in PBS-Triton buffer and once in distilled water, andthen allowed to air dry for 1 hour. The resistance of each element wasthen measured using the same ohmmeter as initially used to measure theresistance of each element.

All elements tested had a starting resistance greater than 2 millionohms. Positive results were obtained for samples at line widths of 3, 5,6 and 7.5 μm. For these samples, the measured resistance at the secondmeasurement, following the reaction, dropped to values ranging from 85to 700 ohms. A few IgG samples resulted in weakly positive resultsranging from 80,000 to 800,000 ohms. Negative results were obtained forthe BSA samples (the resistance after the reaction remained higher than2 million ohms), except that two weakly positive results for BSA sampleswere obtained at the 5 μm line size.

Similar results were obtained when the foregoing procedure was repeatedat different temperatures or incubation times. Usually, a positiveresult was obtained with a reaction time of 20 minutes. These variationsof the procedure indicate that the reaction time can be shortenedsubstantially.

EXAMPLE 2 A. Preparation of protein-labelled colloidal gold anddiagnostic elements

Protein labelled colloidal gold was prepared as described in Example 1,parts A and B. A diagnostic element according to the invention was thenprepared by first coating a glass microscope slide with a thin layer ofchromium by rf magnetron sputter deposition using the procedure ofExample 1, part C, except that no intervening layer of chromium oxidewas formed. The chromium-coated slide was then subjected tophotolithography as described in Example 1, part D, except that the lineformed had a width of 1 millimeter.

A thin layer of carbon was then deposited over the entire upper surfaceof the slide, as shown in FIG. 6, by sputter deposition according toExample 1, part C, except as follows: the backfill gas was argon, andthe target (cathode) was a graphite disk 8 inches in diameter and 0.25inch thick. The graphite disk was epoxyed to a water-cooled copperbacking plate. Deposition was carried out at 145 V with a forward powerof 200 W. The desired carbon layer having a thickness of about 0.25 μmwas thereby formed by deposition in the argon plasma.

B. Measurement of Immunoloqic Reaction

The resistance of the diagnostic elements prepared in part A wasmeasured using an ohmmeter. Each element was then placed in a test tubeand rinsed with a coating buffer (pH 9.8, as described above). Pairs ofpositive and negative diagnostic elements were prepared by adding eitherrabbit immunoglobulin (IgG) or bovine serum albumin (BSA) to each testtube. The elements were incubated by allowing the tubes to standovernight at room temperature. The protein-coated elements were thenwashed three times with a PBS-Triton buffer (phosphate buffered salinesolution as described above containing 0.1 vol. % Triton X-100 as awetting agent) to remove excess unbound protein. The coated substateswere then placed on a piece of parafilm in a petri dish. The colloidalgold-protein conjugate was then applied to each element in an amountsufficient to cover the upper surface of each element, and the elementswere allowed to incubate (stand) for 2, 1 or 1/2 hour at roomtemperature. Each element was then washed once in PBS-Triton buffer andonce in distilled water, and then allowed to air dry for 1 hour. Theresistance of each element was then measured using the same ohmmeter asinitially used to measure the resistance of each element.

C. Silver Enhancement

The elements prepared in part B above were rewashed with distilled waterto remove chloride ions that might react with the silver enhancementreagents. A 2 M sodium citrate buffer solution, a 0.5 M hydroquinonesolution and a 0.03 M silver lactate solution were prepared in adarkened room. The three reagents were then mixed together to provide 25ml of overcoating reagent. The elements were completely immersed in thisreagent for 2-3 minutes, then immersed in a 1% acetic acid solution for2 minutes, and then immersed in a fixative solution for 2 minutes. Thefixative used was Kodak Rapid Fix, containing ammonium thiosulfate,acetic acid, sodium metabisulfite, sodium tetraborate, and aluminumsulfate. The elements were rinsed in distilled water for 10-15 minutesand allowed to air dry. The resistance of each element was then measuredusing the same ohmmeter as used in Example 2, part B.

The resistance results (R) are summarized in Table 2. In part B, Samples6A, 6B were incubated for 1 hour, Samples 7A, 7B were incubated for 1/2hour, and the remaining samples were incubated for 2 hours.

                  TABLE 2                                                         ______________________________________                                                Starting R After   R After                                                    R        Gold      Silver    %                                        Sample  (Ohms)   (Ohms)    Enh. (Ohms)                                                                             Change                                   ______________________________________                                        1A (BSA)                                                                              298,000  394,000   304,000   +0.002                                   1B (IgG)                                                                              357,000  444,000   379       -99.9                                    2A (BSA)                                                                              1,060,000                                                                              1,280,000 950,000   -10.0                                    2B (IgG)                                                                              1,110,000                                                                              1,270,000 6,800     -99.1                                    3A (BSA)                                                                              4,900,000                                                                              6,210,000 4,050,000 -17.3                                    3B (IgG)                                                                              5,460,000                                                                              6,560,000 1,410,000 -74.2                                    4A (BSA)                                                                              39,800   71,500    43,500    +9.3                                     4B (IgG)                                                                              38,800   59,300    192       -99.5                                    5A (BSA)                                                                              3,600,000                                                                              3,600,000 2,600,000 -27.8                                    5B (IGG)                                                                              1,100,000                                                                              1,100,000 603       -99.9                                    6A (BSA)                                                                              48,600   85,200    71,000    +46.1                                    6B (IGG)                                                                              72,500   130,300   920       -98.7                                    7A (BSA)                                                                              29,400   50,300    41,400    +40.8                                    7B (IGG)                                                                              42,600   69,400    690       -98.4                                    ______________________________________                                    

An increase in resistance of about 10-100% normally occurs when thecarbon films of Example 2 are exposed to water. At a channel width of 1mm, no complete bridging occurs which might cause a decrease inresistance. However, resistive shunting embodiments according to theinvention can successfully detect a binding reaction even without metalovercoating if a narrow channel width (or shorter path) is used, andother reaction conditions are adjusted accordingly.

D. Ultrasound Procedure

A pair of BSA and IgG diagnostic elements were prepared according to theprocedure of Example 2, part B, using a 2 hour incubation period. After2 hours both surfaces were washed and dried. A high level of goldparticle binding was observed microscopically for the IgG sample, and amoderate to low amount of binding was observed for the BSA sample. Bothsamples were then immersed in distilled water and subjected to 1 minuteof treatment with a Virsonic 50 Virtis cell disrupter at its lowestpower setting. The samples were then removed and reexamined. Nodifference in the amount of gold binding was observed for the IgGsample, whereas most of the previously observed gold particles on theBSA sample were removed by the treatment. In another experiment in whicha similar ultrasonic treatment was used, a false positive sample (a BSAsample for which a drop in resistance was noted) of the type describedin Example 1, part E, was converted to a negative sample.

EXAMPLE 3

The bioreactivity of various substances was measured by the followingprocedure. The surface to be tested was cut to a size of 1 square cm.For metal oxides and nitrides, the procedure of Example 1, part C, wasvaried as needed to prepare thin film-coated glass test samples. Thesquare samples were placed in test tubes. One ml of a coating buffer asdescribed above was added to each tube to fully cover each surface. Thetubes were shaken gently to wash the surfaces, and then the buffer wasremoved by aspiration. One ml of a solution of 5 μg/ml rabbit IgG incoating buffer was added to each tube. One ml of 1% BSA in coatingbuffer was added to each of a duplicate set of tubes as negativecontrols. All tubes were shaken gently, then allowed to incubateovernight at room temperature. The IgG proteins thereby became bound tothe surfaces.

The coating solutions were then removed by aspiration and washed oncewith PBS-Triton solution (described above). The test samples weretransferred to clean test tubes and washed twice more with PBS-Tritonsolution. One ml of goat anti-rabbit IgG antibody-peroxidase conjugatediluted 1:4000 parts by volume in a PBS-G solution (0.01 M sodiumphosohate dibasic, 0.15 M sodium chloride, 0.1% gelatin) was added toeach test tube. The tubes were allowed to incubate for 30 minutes, thenthe conjugate solution was removed by aspiration, and each sample waswashed 3 times with PBS-Triton solution to remove excess unboundantibody. One ml of 2 mg/ml o-phenylenediamine (OPD) in a substratebuffer (0.058 M sodium phosphate dibasic, 0.023 M citric acid, pH 5.6)and containing 4% hydrogen peroxide was added to each tube. The OPD dyewas activated by the antibody-peroxidase conjugate to produce a yellowcolor which absorbs light at 490 nm.

The tubes were incubated for 5 minutes at room temperature, then thereaction was stopped by adding 0.4 ml of 2 M sulfuric acid to each tube,and each tube was mixed. 300 μl of each tube was transferred to a wellof a microtiter plate, and 300 μl of water was also placed in a well asa reference blank. Absorbance measurements for each well were made usinga Bio-Tek Automated Microplate Reader Model EL309. The amount of proteinbound to each sample is directly proportional to the absorbance of thecorresponding sample in Absorbance Units (AU) at 490 nm. The resultswere:

                  TABLE 3                                                         ______________________________________                                        Sample          Binding Ability (%)                                           ______________________________________                                        Nylon 66        100                                                           Polypropylene   90                                                            Mylar           89                                                            Chromium oxide  83                                                            Phenolic plastic                                                                              78                                                            Polystyrene     73                                                            Vinyl           65                                                            Chromium        59                                                            Nickel oxide    52                                                            Styrene         29                                                            Titanium oxide  16                                                            Boron nitride   13                                                            Silicon oxide   10                                                            Silicon nitride 7                                                             Barium titanium oxide                                                                         6                                                             Indium tin oxide                                                                              6                                                             Aluminum oxide  6                                                             Glass           4                                                             ______________________________________                                    

For the BSA controls and the samples for IgG bioinert surfaces such asglass, the antibody-peroxidase conjugate did not bind, so that the OPDdid not change color and the absorbance at 490 nm was low.Correspondingly higher absorption values were obtained by bioactivesubstances tested, such as chromium oxide and Nylon 66. Somewhatdifferent results were obtained when the experiment was repeated usingfour DNA-coated surfaces instead of protein-coated surfaces. Chromiumoxide showed the highest bioreactivity (0.531 AU), followed by glass(0.393 AU), titanium oxide (0.378 AU) and then polystyrene (0.279 AU).Metal oxides thus seem to have a better affinity for nucleotides thanplastics such as polystyrene.

EXAMPLE 4

The sensitivity of the method of the invention was evaluated bydetermining a limiting amount of antibody required for an assay.Diagnostic elements were prepared by cutting conductive polyimideplastic (Kapton containing blended carbon) into 1 cm by 0.3 cm squares.The pieces were rinsed once in coating buffer (described above) andimmersed overnight at room temperature in test tubes containing either1% BSA in coating buffer or 5 μg/ml rabbit IgG (the antigen). Theresulting sample elements were then washed three times with PBS-Tritonsolution (described above) and placed on parafilm in petri dishes. Asolution of goat anti-rabbit IgG antibody bound to gold particles,prepared according to the procedure of Example 1, Parts A and B, wasserially diluted, and two drops at each dilution were applied to oneBSA-coated sample and one IgG-coated sample. After 2 hours of incubationat room temperature, the samples were first washed with PBS-Tritonsolution, and then with distilled water. The samples were then allowedto dry, and the resistance of each sample was measured. Silverovercoating was then carried out for each sample using the procedure ofExample 2, part C. The resistance of each sample was then measured, andthe number of silver counts at each dilution was determined by x-rayanalysis.

The results were as follows, wherein resistance values (R) are given inmegaohms except where noted:

                  TABLE 4                                                         ______________________________________                                                              R After  R After Silver                                 Sample/Dilution                                                                           Starting R                                                                              Gold     Silver  Counts                                 ______________________________________                                        1A  (BSA)   None    1.55    2.02   1.46      --                               1B  (IgG)   None    1.48    1.72   112  ohms 2861                             2A  (BSA)   1:2     1.52    2.11   1.28      --                               2B  (IgG)   1:2     1.47    --     --        2187                             3A  (BSA)   1:4     1.52    1.94   1.67      --                               3B  (IgG)   1:4     1.53    1.57   0.31      1723                             4A  (BSA)   1:8     1.41    1.60   1.43      --                               4B  (IgG)   1:8     1.41    1.57   0.54      1174                             5A  (BSA)    1:16   1.40    1.58   1.41      --                               5B  (IgG)    1:16   1.49    1.57   0.62       748                             6A  (BSA)    1:32   1.48    1.85   1.46      --                               6B  (IgG)    1:32   1.47    1.82   1.06       699                             ______________________________________                                    

Resistance values after silver overcoating and silver counts obtained inthis example are plotted against gold-antibody conjugate dilution inFIG. 13, wherein X's represent silver counts and circles representresistance in megaohms. The limiting antibody concentration was found tooccur at a dilution of about 1:8. The resistance and silver count valuesboth varied substantially linearly with concentration, and the change inresistance was found to vary inversely with the number of silver counts,demonstrating that the extent of resistive shunting was directly relatedto the number of silver overcoated aggregates bound to the surface.

EXAMPLE 5

The sensitivity of the method of the invention was further evaluated bygenerating a dose response curve. Diagnostic elements were prepared bycutting conductive Kapton plastic as described in Example 4 into 2.3 cmby 0.3 cm pieces. Titanium metal contact pads (0.5 by 0.3 cm) weredisposed at opposite ends of each strip 1.3 cm apart to form resistors.The resistance of each resistor was then measured. The resistors wererinsed once with coating buffer, incubated in rabbit IgG, and washedthree times using the procedure of Example 4. Dilutions of rabbit IgG incoating buffer containing rabbit IgG in the amounts given in Table 5below were prepared and preincubated with gold-goat anti-rabbit IgGconjugate prepared according to Example 4 at a 1:8 dilution for 11.5minutes. The preincubated conjugate was then applied to the sampleresistors disposed on parafilm in petri dishes and allowed to incubatefor 2 hours at room temperature. The samples were then washed once withPBS-Triton solution and once with distilled water, and allowed to dry.The resistance of each sample was then measured by connecting anohmmeter to each of the contact pads. The samples were then silverovercoated as described in Example 2, part C, and the resistance of thesamples was again measured.

The results were as follows, wherein the resistance values (R) areexpressed in kiloohms and the amount of rabbit IgG (antigen) isexpressed in nanograms:

                  TABLE 5                                                         ______________________________________                                               Rab.IgG            R After                                                                              R After                                                                              %                                     Sample Amount   Starting R                                                                              Gold   Silver Change                                ______________________________________                                        1A     1000     311       349    335    -4.0                                  1B     1000     318       355    340    -4.2                                  2A     500      391       410    395    -3.7                                  2B     500      347       375    351    -6.4                                  3A     100      347       383    344    -10.2                                 3B     100      350       410    326    -20.5                                 4A      50      371       406    371    -8.6                                  4B      50      376       416    385    -7.5                                  5A      10      388       434    368    -15.2                                 5B      10      370       444    399    -10.1                                 6A      5       437       515    443    -14.0                                 6B      5       394       498    394    -20.9                                 7A      1       418       498    178    -64.3                                 7B      1       392       480     99    -79.4                                 ______________________________________                                         FIG. 14 graphically depicts a plot of percent of maximum resistance (100%     minus percent change from Table 5) versus the amount of competing antigen,     using the average of the two results A,B obtained for each amount of     antigen plotted. The results indicate that the method of this example was     sensitive enough to detect as little as 1 nanogram of antigen in a sample.

The procedures set forth in the foregoing examples may also be used toprepare other useful devices which need to have a bioreactive surface,for example, a bioimplant medical device such as an artificial heart orprosthetic part. In particular, the biocompatability of a metal implantcan be improved by providing it with a thin coating of a bioreactivesubstance according to the invention, such as bioreactive plastic orchromium oxide. A layer of the recipient's own tissue may then bind tothe bioreactive layer. The resulting device has a surface which can bindmore firmly to the surrounding tissue and which is less likely to berejected.

It will be understood that the above description is of preferredexemplary embodiments of the present invention, and the invention is notlimited to the specific forms shown. The method of the invention is notlimited to measuring or detecting an unknown quantity of a substance ina patient sample. It can also be used in research when it is desired tomeasure features of a binding reaction between two known substances, forexample, the speed or extent of the reaction, or to construct a table ofstandard values for later use in analyzing a patient sample. These andother modifications may be made in the design of the invention withoutdeparting from the scope of the present invention as expressed in theappended claims.

We claim:
 1. A method of detecting a substance in a test sample, which substance is a first one of a pair of first and second substances that undergo a specific binding reaction with each other, including the steps of:mixing the sample with particles under conditions effective to cause binding of the first substance, if present in the sample, to the surfaces of the particles; contacting the particles having the first substance bound to the surfaces thereof with a layer of the second of the pair of substances, which layer forms a path between a pair of spaced-apart electrical conductors superposed on a substantially nonelectrically conductive base, the layer of the second substance being bound to the base, such that the binding reaction between the first and second substances causes the particles to be bound to the path in aggregates; removing particles which are bound to the path as a result of non-specific binding and particles which are unbound to the path; coating exposed outer surfaces of the aggregates with an electrically conductive substance that adheres selectively to the particles forming the aggregates but which does not adhere to the remainder of the path; removing electrically conductive substances which remains unadhered to the aggregates; connecting each of the conductors to an electrical circuit which includes a source of electrical energy, the conductors and the path therein; and measuring a change in the electrical current flow through the circuit caused by formation of the coated aggregates on the path, the electrical change indicating the level of the substance to be detected in the test sample.
 2. The method of claim 1, wherein the step of measuring a change in current flow comprises measuring a change in the electrical resistance across the electrical circuit.
 3. The method of claim 1, wherein the step of coating the aggregates comprises treating the aggregates with a silver compound-containing coating reagent, treating the silver coated aggregates with an acid solution, fixing the silver coating, and rinsing the coated aggregates.
 4. The method of claim 1, wherein the step of removing unbound particles comprises ultrasonically treating non-specifically bound particles and washing the path.
 5. The method of claim 1, wherein the pair of substances comprises an antibody and an antigen.
 6. The method of claim 1, wherein the particles are selected from particles of gold, silver, platinum, carbon, or conductive metal-coated plastic, and the electrically conductive substance is a metal.
 7. The method of claim 6, wherein the conductors comprise a pair of conductive layers made of a conductive metal disposed side-by-side on the base, the path lying in a channel between the conductive layers, and the base has a bioreactive surface such that the layer of the second substance reacts and becomes bound thereto and comprises a support having a surface made of a bioreactive material selected from carbon, hydrophilic organic polymers, inorganic metal oxides and inorganic metal nitrides.
 8. The method of claim 7, wherein the channel has a width of up to 1 cm, and the conductive layers have a thickness of less than 0.5 micron and are made of a metal selected from gold, silver, platinum, copper, chromium and aluminum.
 9. The method of claim 8, wherein the bioreactive material is carbon.
 10. The method of claim 1, wherein one member of the specific binding pair is selected from the group consisting of hormones, allergens, nucleotides, and enzymes.
 11. A method of detecting a substance in a test sample, which substance is a first one of a pair of first and second substances that undergo a specific binding reaction with each other, including the steps of:mixing the sample with particles having the second one of the substances bound to the surfaces thereof under conditions effective to cause binding of the first substance, if present in the sample, to the second substance on the surfaces of the particles; contacting the particles with a layer of the first substance, which layer forms a path between a pair of spaced-apart electrical conductors superposed on a substantially nonelectrically conductive base, the layer of the first substance being bound to the base, such that the binding reaction between the first and second substances causes the particles to be bound to the path in aggregates, and particles on which the second substance became bound to the first substance from the test sample in sufficient quantity do not become bound to the path; removing particles which are bound to the path as a result of non-specific binding and particle which are unbound to the path; coating exposed outer surfaces of the aggregates with an electrically conductive substance that adheres selectively to the particles forming the aggregates but which does not adhere to the remainder of the path; removing electrically conductive substance which remains unadhered to the aggregates; connecting each of the conductors to an electrical circuit which includes a source of electrical energy, the conductors and the path therein; and measuring a change in the electrical current flow through the circuit caused by formation of the coated aggregates on the path, the electrical change indicating the level of the substance to be detected in the test sample.
 12. The method of claim 11, wherein the step of measuring a change in current flow comprises measuring a change in the electrical resistance across the electrical circuit.
 13. The method of claim 11, wherein the step of coating the aggregates comprises treating the aggregates with a silver compound-containing coating reagent, treating the silver coated aggregates with an acid solution, fixing the silver coating, and rinsing the coated aggregates.
 14. The method of claim 11, wherein the step of removing unbound particles comprises ultrasonically treated non-specifically bound particles and washing the path.
 15. The method of claim 11, wherein the pair of substances comprises an antibody and an antigen.
 16. The method of claim 11, wherein the particles are selected from particles of gold, silver, platinum, carbon, or conductive metal-coated plastic, and the electrically conductive substance is a metal.
 17. The method of claim 16, wherein the conductors comprises a pair of conductive layers made of a conductive metal disposed side-by-side on the base, the path lying in a channel between the conductive layers, and the base has a bioreactive surface such that the layer of the second substance reacts and becomes bound thereto and comprises a support having a surface made of a bioreactive material selected from carbon, hydrophilic organic polymers, inorganic metal oxides and inorganic metal nitrides.
 18. The method of claim 17, wherein the channel has a width of up to 1 cm, and the conductive layers have a thickness of less than 0.5 micron and are made of a metal selected from gold, silver, platinum, copper, chromium and aluminum.
 19. The method of claim 18, wherein the bioreactive material is carbon.
 20. The method of claim 11, wherein on member of the specific binding pair is selected from the group consisting of hormones, allergens, nucleotides, and enzymes. 